Enhancement of Electron Spin Echo Envelope Modulation Spectroscopic Methods to Investigate the Secondary Structure of Membrane Proteins

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• 作者：Lishan Liu ; Indra D. Sahu ; Daniel J. Mayo ; Robert M. McCarrick ; Kaylee Troxel ; Andy Zhou ; Erin Shockley ; Gary A. Lorigan
• 刊名：The Journal of Physical Chemistry B
• 出版年：2012
• 出版时间：September 13, 2012
• 年：2012
• 卷：116
• 期：36
• 页码：11041-11045
• 全文大小：225K
• 年卷期：v.116,no.36(September 13, 2012)
• ISSN：1520-5207

文摘

This paper reports on a significant improvement of a new structural biology approach designed to probe the secondary structure of membrane proteins using the pulsed EPR technique of electron spin echo envelope modulation (ESEEM) spectroscopy. Previously, we showed that we could characterize an 伪-helical secondary structure with ESEEM spectroscopy using a ²H-labeled Val side chain coupled with site-directed spin-labeling (SDSL). In order to further develop this new approach, molecular dynamic (MD) simulations were conducted on several different hydrophobic residues that are commonly found in membrane proteins. ²H-SL distance distributions from the MD results indicated that ²H-labeled Leu was a very strong candidate to significantly improve this ESEEM approach. In order to test this hypothesis, the secondary structure of the 伪-helical M2未 peptide of the acetylcholine receptor (AChR) incorporated into a bicelle was investigated with ²H-labeled Leu d₁₀ at position 10 (i) and nitroxide spin labels positioned 1, 2, 3, and 4 residues away (denoted i+1 to i+4) with ESEEM spectroscopy. The ESEEM data reveal a unique pattern that is characteristic of an 伪-helix (3.6 residues per turn). Strong ²H modulation was detected for the i+3 and i+4 samples, but not for the i+2 sample. The ²H modulation depth observed for ²H-labeled d₁₀ Leu was significantly enhanced (脳4) when compared to previous ESEEM measurements that used ²H-labeled d₈ Val. Computational studies indicate that deuterium nuclei on the Leu side chain are closer to the spin label when compared to Val. The enhancement of ²H modulation and the corresponding Fourier Transform (FT) peak intensity for ²H-labeled Leu significantly reduces the ESEEM data acquisition time for Leu when compared to Val. This research demonstrates that a different ²H-labeled amino acid residue can be used as an efficient ESEEM probe further substantiating this important biophysical technique. Finally, this new method can provide pertinent qualitative structural information on membrane proteins in a short time (few minutes) at low sample concentrations (50 渭M).